Immunoprotective primary mesenchymal stem cells and methods

ABSTRACT

Immunoprotective primary mesenchymal stems cells (IP-MSC) which episomally express immunoreactive polypeptides that specifically target a pathogen (e.g., an infectious species of virus, bacterium, or parasite) or toxin are described herein. The immunoreactive polypeptides can be, e.g., full antibodies, single-chain antibodies (ScFV), Fab or F(ab) 2  antibody fragments, diabodies, tribodies, and the like). Optionally IP-MSC are transfected to express one or more other immunomodulating polypeptides, e.g., a cytokine such as an interleukin (e.g., IL-2, IL-4, IL-6, IL-7, IL-9, and IL-12), an interferon (e.g., IFNα, IFNβ, or IFNω), and the like, which can enhance the effectiveness of the immunoreactive polypeptides.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.14/802,247, filed on Jul. 17, 2015, which is a continuation of U.S.application Ser. No. 13/826,285, filed on Mar. 14, 2013, now U.S. Pat.No. 9,101,587, each of which is incorporated herein by reference in itsentirety.

FIELD OF THE INVENTION

This invention relates to mesenchymal stems cells. More particularly,this invention relates to primary mesenchymal stems cells (MSC) fordelivery of polypeptides that are immunoreactive against pathologicalagents such as pathogens and toxins, as well as methods for preparationof such MSC and deployment of such MSC against pathological agents.

SEQUENCE LISTING INCORPORATION

Biological sequence information for this application is included in anASCII text file having the file name “TU-491-SEQ.txt”, created on Mar.11, 2013, and having a file size of 12,954 bytes, which is incorporatedherein by reference.

BACKGROUND

Mesenchymal stem cells (MSC) are unique multipotent progenitor cellsthat are presently being exploited as gene therapy vectors for a varietyof conditions, including cancer and autoimmune diseases. Although MSCare predominantly known for anti-inflammatory properties duringallogeneic MSC transplant, there is evidence that MSC can actuallypromote adaptive immunity under certain settings. MSC have beenidentified in a wide variety of tissues, including bone marrow, adiposetissue, placenta, and umbilical cord blood. Adipose tissue is one of therichest known sources of MSC.

MSC have been successfully transplanted into allogeneic hosts in avariety of clinical and pre-clinical settings. These donor MSC oftenpromote immunotolerance, including the inhibition of graft-versus-hostdisease (GvHD) that can develop after cell or tissue transplantationfrom a major histocompatibility complex (MHC)-mismatched donor. Thediminished GvHD symptoms after MSC transfer has been due to direct MSCinhibition of T and B cell proliferation, resting natural killer cellcytotoxicity, and dendritic cell (DC) maturation. At least one study hasreported generation of antibodies against transplanted allogeneic MSC.Nevertheless, the ability to prevent GvHD also suggests that MSCexpressing foreign antigen might have an advantage over other cell types(i.e., DC) during a cellular vaccination in selectively inducing immuneresponses to only the foreign antigen(s) expressed by MSC and notspecifically the donor MSC.

The use of modified MSC also has been explored in vivo in order toenhance the immunomodulatory properties of MSC. MSC transduced tooverproduce IL-10 suppressed collagen-induced arthritis in a mouse model(Choi et al., 2008). In addition, MSC expressing glucagon-like peptide-1transplanted into an Alzheimer's disease mouse model led to a decreasein A-beta deposition in the brain (Klinge et al., 2011). In anosteopenia mouse model, mice receiving transduced MSC that had stableoverexpression of bone morphogenetic protein had increased bone density(Kumar et al., 2010). In a rat model for spinal cord injury, ratstreated with MSC stably overexpressing brain-derived neurotrophic factorhad a better overall outcome than rats administered MSC alone (Sasaki etal., 2009). Lastly, in a rat model for bladder outlet obstruction, ratsreceiving transduced MSC with stable overexpression of hepatocyte growthfactor had decreased collagen accumulation in the bladder (Song et al.,2012). These studies indicate that modified MSC are a useful andfeasible vehicle for protein expression and delivery to target variousdiseases and tissues.

MSC have been studied as a delivery vehicle for anti-cancer therapeuticsdue to their innate tendency to home to tumor microenvironments, and isthoroughly reviewed in (Loebinger and Janes, 2010). MSC also have beenused to promote apoptosis of tumorigenic cells through the expression ofIFNα or IFNγ (Li et al., 2006; Ren et al., 2008). Additionally, MSCrecently have been explored for the prevention and inhibition oftumorigenesis and metastasis. A study by Wei et al. examined the use ofhuman papilloma virus (HPV)-immortalized MSC that express the HPVproteins E6/E7 combined with a modified E7 fusion protein vaccine in amouse tumor model where metastatic fibrosarcoma cells were administered(Wei et al., 2011). This group found that only mice that were immunizedwith both the E7-expressing MSC and modified E7 protein vaccine showed adecrease in tumor growth, and an E7-specific antibody response. Micereceiving either MSC or protein vaccine alone were not able to raise ananti-E7 response or inhibit tumor growth of metastatic sarcoma. Althoughthese immortalized MSC were previously determined to be non-tumorigenic,they persisted in mice longer than 21 days, unlike primary MSC (i.e.non-immortalized), which are only detectable for a very short time afteradministration (Gao et al., 2001; Abraham et al., 2004; Ohtaki et al.,2008; Prockop, 2009). Thus, there may be unforeseen outcomes in the longterm (i.e., outcompeting with endogenous MSC and differingimmunomodulatory abilities, which were not assessed in this study) withthe use of immortalized MSC, even if they prove to be non-malignant.Other studies have indicated that immortalized MSC can becometumorigenic, and thus must be carefully studied to determine if they areindeed safe for use. Transplanted primary non-immortalized MSC persistonly for a few days at most in vivo (Gao et al., 2001; Abraham et al.,2004; Ohtaki et al., 2008; Prockop, 2009).

While MSC are primarily touted for their immunosuppressive properties,several published reports have also directly shown that MSC promoteadaptive immunity. In co-cultures, MSC enhanced B-cell proliferation,IL-6 expression and IgG-secreting plasma cell formation in vitro; theseB-cell responses could be further augmented with MSC combined with a TLRagonist (lipopolysaccharide or CpG DNA). MSC pulsed with tetanus toxoidpromoted the proliferation and cytokine expression (IL-4, IL-10, IFNγ)of a tetanus toxoid-specific CD4 T-cell line. Similarly, MSC cultured inlow ratios (1:100) with lymphocytes in the presence of antigen improvedlymphocyte proliferation and CD4 Th17 subset formation, which waspartially IL-6 and TGFβ-dependent. MSC have also been found to expressMHC-I and cross-present antigen for expansion of CD8 T-cells both invitro and in vivo.

MSC immunoregulation has also been found to be dependent upon externalsignals. In the presence of inflammatory cytokines or stimulants, MSCtherapy, which was previously suppressive, can become immunostimulatory.For example, MSC treated with specific pathogen-associated molecularpattern (PAMP) molecules can become either anti- or pro-inflammatory,depending on the PAMP with which they are treated in vitro. Duringcollagen-induced arthritis, an inflammatory disease setting,transplantation of allogeneic MSC reportedly enhanced Th1 immuneresponses and IL-6 secretion, which was mimicked in vitro by direct TNFαstimulation of MSC. Administration of MSC also reportedly exacerbatedcollagen-induced arthritis disease and amplified splenocyte secretion ofIL-6 and IL-17. Pre-treatment of MSC with IFNγ (within a moderate range)reportedly upregulates MHC-I and II expression and improves antigenphagocytosis and presentation capabilities, thereby stimulating CD4 andCD8 T-cell proliferation and generation of anti-tumor CD8+ cytotoxicT-lymphocytes (CTLs).

Vaccines often are efficient and cost-effective means of preventinginfectious disease. Vaccines have demonstrated transformative potentialin eradicating one devastating disease, smallpox, while offering theability to control other diseases, including diphtheria, polio, andmeasles, that formerly caused widespread morbidity and mortality. Thedevelopment of vaccines involves the testing of an attenuated orinactivated version of the pathogen or identification of a pathogencomponent (i.e., subunit, toxoid, and virus-like particle vaccines) thatelicits an immune response that protects recipients from disease whenthey are exposed to the actual pathogen. In an ideal world a singlevaccine would be able to target all major human pathogens (versatile),elicit strong protective immunity to these pathogens without inducingunwanted side-effects, and still be fairly inexpensive to produce perdose. In the case of viruses or host-cell produced proteins, vaccineproduction that includes human post-translational processing, mimickingnatural infection, will likely prove to be superior to bacterial orother expression systems.

Traditional vaccine approaches have thus far failed to provideprotection against HIV, tuberculosis, malaria and many other diseases,including dengue, herpes and even the common cold. The reasons whytraditional vaccine approaches have not been successful for thesediseases are complex and varied. For example, HIV integrates functionalproviral genomes into the DNA of host cells, thereby establishinglatency or persistence. Once latency/persistence is established, it hasnot been possible to eradicate HIV, even with highly activeantiretroviral therapy.

Newer alternative immunization approaches include both DNA and cellularvaccines. DNA vaccines involve the transfection of cells at the tissuesite of vaccination with an antigen-encoding plasmid that allows localcells (i.e. myocytes) to produce the vaccine antigen in situ. Cellularvaccines use the direct transfer of pre-pulsed or transfected hostantigen presenting cells (e.g., dendritic cells, DC) expressing orpresenting the vaccine antigen. The advantage of these approaches isthat vaccine antigens are produced in vivo and are readily available forimmunological processing. Despite numerous reports of successfulpre-clinical testing, both such approaches have hit stumbling blocks.DNA vaccination studies in humans show poor efficacy, which has beenlinked to innate differences between mice and humans (Cavenaugh et al.,2011; Wang et al., 2011). DC vaccination strategies have shown limitedclinical success for therapeutic cancer vaccinations and have highproduction costs due to necessary individual tailoring (Bhargava et al.,2012; Palucka and Banchereau, 2012).

A further limitation on current vaccine technology is the time involvedin developing a vaccine against a give pathogen. This is particularlyproblematic in the case of exposure to newly emerging pathogens anddeliberately or accidentally released pathogens and toxins, where themeans for rapid protection to contain such emerging pathogens andbiological threats are needed. The methods and episomally transfectedMSC described herein address these needs.

SUMMARY OF THE INVENTION

The present invention provides immunoprotective primary mesenchymalstems cells (IP-MSC), which episomally express multiple immunoreactivepolypeptides that specifically target a pathogen (e.g., an infectiousspecies of virus, bacterium, or parasite) or a toxin, as well as methodsof preparing and using the IP-MSC. The IP-MSC are transfected with oneor more episomal vectors encoding two or more (e.g., 2 to about 100)expressible immunoreactive polypeptides (e.g., full antibodies, singlechain variable antibodies fragments (ScFV), Fab or F(ab′)₂ antibodyfragments, diabodies, tribodies, and the like). Optionally, the IP-MSCcan express one or more other immunomodulating polypeptides, e.g., acytokine such as an interleukin (e.g., IL-2, IL-4, IL-6, IL-7, IL-9, andIL-12), an interferon (e.g., IFNα, IFNβ, or IFNω), and the like, whichcan enhance the effectiveness of the antigen-binding polypeptides toneutralize the pathogen or toxin. Each immunoreactive polypeptidecomprises an amino acid sequence of an antigen-binding region from or ofa neutralizing antibody (e.g., a native antibody from an exposedsubject) specific for an antigen produced by the pathogen or specificfor the toxin, or comprises an amino acid sequence of a variant of theantigen-binding region that includes one or more substitutions (e.g.,conservative substitutions) in the amino acid sequence thereof, andpreferably sharing at least about 50% sequence identity (e.g., at leastabout 60, 70, 80, 90, or 95% sequence identity) with the nativeantigen-binding region. Each antigen-binding region peptide or variantthereof is arranged and oriented to specifically bind to and neutralizethe pathogen or toxin.

In some embodiments the IP-MSC express, e.g., at least 2, 3, 4, 5, or 6immunoreactive polypeptides, or up to about 10, 20, 30, 40, 50, 60, 70,80, 90 or 100 immunoreactive polypeptides, which specifically target thepathogen or toxin. For example, each immunoreactive polypeptide canspecifically target and bind to a protein or fragment thereof from apathogenic organism, or to a toxin (e.g., ricin, abrin, anthrax toxin,botulinium toxin), which can be produced by an organism in situ or maybe encountered in a chemically isolated or purified form.

The IP-MSC are useful for generating passive immunity against ortreating an infection by the pathogen or exposure to the toxin (e.g., byneutralization). The IP-MSC can be provided in a pharmaceuticallyacceptable carrier (e.g., a buffer, such as phosphate buffered saline,or any other buffered material suitable for sustaining viabletransfected primary MSC) for use as a pharmaceutical composition fortreating or preventing an infectious disease caused by the pathogen orameliorating deleterious effects of a toxin. In some embodiments, theIP-MSC comprise bone-marrow derived MSC, while in some otherembodiments, the IP-MSC comprise adipose MSC cells, placental MSC cells,or umbilical cord blood MSC cells.

The IP-MSC described herein are particularly useful for temporarypassive protection against pathogens and toxins, at least in part,because primary MSC are hypo-immunogenic cells that generally are nottargeted by the immune system. Thus, the IP-MSC are tolerated by thetreated subject, allowing the cells to survive for a sufficient time forimmunoreactive polypeptides to be expressed, produced, and released tobind to and neutralize a pathogenic organism or toxin to which thesubject has been or may be exposed. In addition, primary MSC generallyhave a limited lifetime in the body, thus ameliorating potential forundesirable long-term side effects of treatment with the MSC (e.g.,carcinogenicity), which may be an issue with immortalized MSC.

The following embodiments 1 to 35 of the present invention are providedto further illustrate the scope and various aspects of the invention.These embodiments are provided as non-limiting illustrations of theIP-MSC and methods described herein.

Embodiment 1 comprises immunoprotective primary mesenchymal stems cells(IP-MSC) that episomally express multiple immunoreactive polypeptidesthat specifically target a pathogen or toxin. The IP-MSC are transfectedwith one or more episomal vectors encoding the immunoreactivepolypeptides. Each immunoreactive polypeptide comprises an amino acidsequence from an antigen-binding region of a neutralizing antibodyspecific for an antigen produced by the pathogen or specific for thetoxin, or an amino acid sequence of a variant of the antigen-bindingregion sequence comprising one or more substitutions in the amino acidsequence thereof and sharing at least about 50% sequence identity withthe sequence of the antigen-binding region; and the antigen-bindingregion sequence or variant thereof is arranged and oriented tospecifically bind to and neutralize the pathogen or toxin; and whereinthe episomal vector optionally includes an inducible apoptosis gene.

Embodiment 2 comprises the IP-MSC of embodiment 1 wherein the IP-MSCepisomally express 2 to about 100 of the immunoreactive polypeptides.

Embodiment 3 comprises the IP-MSC of embodiment 1 or 2 wherein theIP-MSC also express one or more other immunomodulating agents.

Embodiment 4 comprises the IP-MSC of embodiment 3 wherein the one ormore immunomodulating agents are selected from interleukins andinterferons.

Embodiment 5 comprises the IP-MSC of embodiment 3 wherein the one ormore immunomodulating agents are selected from IL-2, IL-4, IL-6, IL-7,IL-9, IL-12, IFNα, IFNβ, and IFNω.

Embodiment 6 comprises the IP-MSC of any one of embodiments 1 to 5wherein each immunoreactive polypeptide is selected from a full-lengthantibody, a single-chain variable antibody fragment (ScFV), a monovalentantibody antigen-binding fragment (Fab), a divalent antibodyantigen-binding fragment (F(ab′)₂), a diabody, and a tribody.

Embodiment 7 comprises the IP-MSC of any one of embodiments 1 to 6wherein one or more of the immunoreactive polypeptides comprises anamino acid sequence of the variant of the antigen-binding region, andwherein the amino acid sequence substitutions of the variant compriseconservative substitutions.

Embodiment 8 comprises the IP-MSC of any one of embodiments 1 to 7wherein one or more of the immunoreactive polypeptides comprises anamino acid sequence of the variant of the antigen-binding region, andwherein the amino acid sequence of the variant shares at least about 80%sequence identity with the sequence of the antigen-binding region.

Embodiment 9 comprises the IP-MSC of any one of embodiments 1 to 8wherein the pathogen is a viral pathogen.

Embodiment 10 comprises the IP-MSC of any one of embodiments 1 to 8wherein the pathogen is a bacterial pathogen.

Embodiment 11 comprises the IP-MSC of any one of embodiments 1 to 8wherein the pathogen is a single-celled parasitic pathogen.

Embodiment 12 comprises the IP-MSC of any one of embodiments 1 to 8wherein the pathogen is a multicellular parasitic pathogen.

Embodiment 13 comprises the IP-MSC of any one of embodiments 1 to 8wherein the pathogen is a viral pathogen selected from the groupconsisting of: an adenovirus; a papillomavirus; a hepadnavirus; aparvovirus; a pox virus; Epstein-Barr virus; cytomegalovirus (CMV); aherpes simplex virus; roseolovirus; varicella zoster virus; a filovirus;a paramyxovirus; an orthomyxovirus; a rhabdovirus; an arenavirus; acoronavirus; a human enterovirus; hepatitis A virus; a human rhinovirus;polio virus; a retrovirus; a rotavirus; a flavivirus; a hepacivirus; andrubella virus.

Embodiment 14 comprises the IP-MSC of any one of embodiments 1 to 8wherein the pathogen is a bacterial pathogen from a genus selected fromthe group consisting of: Bacillus; Bordetella; Borrelia; Brucella;Burkholderia; Campylobacter; Chlamydia, Chlamydophila; Clostridium;Corynebacterium; Enterococcus; Escherichia; Francisella; Haemophilus;Helicobacter; Legionella; Leptospira; Listeria; Mycobacterium;Mycoplasma; Neisseria; Pseudomonas; Rickettsia; Salmonella; Shigella;Staphylococcus; Streptococcus; Treponema; Vibrio; and Yersinia.

Embodiment 15 comprises the IP-MSC of any one of embodiments 1 to 8wherein the pathogen is a parasitic pathogen selected from the groupconsisting of: Acanthamoeba; Anisakis; Ascaris lumbricoides; Balantidiumcoli; Cestoda (tapeworm); Chiggers; Cochliomyia hominivorax; Entamoebahistolytica; Fasciola hepatica; Giardia lamblia; Hookworm; Leishmania;Linguatula serrata, Liver fluke; Loa loa; Paragonimus (lung fluke);Pinworm; Plasmodium falciparum; Schistosoma; Strongyloides stercoralis;Tapeworm; Toxoplasma gondii; Trypanosoma; Whipworm; and Wuchereriabancrofti.

Embodiment 16 comprises the IP-MSC of any one of embodiments 1 to 8wherein the antigenic polypeptide is selected from the group consistingof: influenza hemagglutinin 1 (HA1); influenza hemagglutinin 2 (HA2);influenza neuraminidase (NA); Lassa virus (LASV) glycoprotein 1 (gp1);LASV glycoprotein 2 (gp2); LASV nucleocapsid-associated protein (NP);LASV L protein; LASV Z protein; SARS virus S protein; Ebola virus GP2;measles virus fusion 1 (F1) protein; HIV-1 transmembrane (TM) protein;HIV-1 glycoprotein 41 (gp41); HIV-1 glycoprotein 120 (gp120); hepatitisC virus (HCV) envelope glycoprotein 1 (E1); HCV envelope glycoprotein 2(E2); HCV nucleocapsid protein (p22); West Nile virus (WNV) envelopeglycoprotein (E); Japanese encephalitis virus (JEV) envelopeglycoprotein (E); yellow fever virus (YFV) envelope glycoprotein (E);tick-borne encephalitis virus (TBEV) envelope glycoprotein (E);hepatitis G virus (HGV) envelope glycoprotein 1 (E1); respiratorysynctival virus (RSV) fusion (F) protein; herpes simplex virus 1 (HSV-1)gD protein; HSV-1 gG protein; HSV-2 gD protein; HSV-2 gG protein;hepatitis B virus (HBV) core protein; Epstein-Barr virus (EBV)glycoprotein 125 (gp125); bacterial outer membrane protein assemblyfactor BamA; bacterial translocation assembly module protein TamA;bacterial polypeptide-transport associated protein domain protein;bacterial surface antigen D15; anthrax protective protein; anthraxlethal factor; anthrax edema factor; Salmonella typhii S1Da; Salmonellatyphii S1Db; cholera toxin; cholera heat shock protein; Clostridiumbotulinum antigen S; botulinum toxin; Yersina pestis F1; Yersina pestisV antigen; Yersina pestis YopH; Yersina pestis YopM; Yersina pestisYopD; Yersina pestis plasminogen activation factor (Pla); Plasmodiumcircumsporozoite protein (CSP); Plasmodium sporozoite surface protein(SSP2/TRAP); Plasmodium liver stage antigen 1 (LSA1); Plasmodiumexported protein 1 (EXP 1); Plasmodium erythrocyte binding antigen 175(EBA-175); Plasmodium cysteine-rich protective antigen (cyRPA);Plasmodium heat shock protein 70 (hsp70); Schistosoma Sm29; andSchistosoma signal transduction protein 14-3-3.

Embodiment 17 comprises the IP-MSC of any one of embodiments 1 to 16wherein the IP-MSC are prepared from bone marrow-derived mesenchymalstem cells.

Embodiment 18 comprises the IP-MSC of any one of embodiments 1 to 16wherein the IP-MSC are prepared from adipose-derived mesenchymal stemcells.

Embodiment 19 comprises the IP-MSC of any one of embodiments 1 to 18 fortreating an infection by the pathogen or toxicity from exposure to atoxin.

Embodiment 20 comprises the IP-MSC of any one of embodiments 1 to 18 forpreventing an infection by the pathogen or preventing toxicity fromexposure to the toxin.

Embodiment 21 comprises a pharmaceutical composition for treating aninfection caused by the pathogen or treating exposure to the toxincomprising the IP-MSC of any one of embodiments 1 to 20 in apharmaceutically acceptable carrier.

Embodiment 22 comprises a pharmaceutical composition for preventing aninfection caused by the pathogen or for ameliorating the effects ofexposure to the toxin comprising the IP-MSC of any one of embodiments 1to 20 in a pharmaceutically acceptable carrier.

Embodiment 23 comprises use of the IP-MSC of any one of embodiments 1 to20 for prevention of an infection caused by the pathogen or preventingtoxicity from exposure to the toxin.

Embodiment 24 comprises use of the IP-MSC of any one of embodiments 1 to20 for treating an ongoing infection caused by the pathogen or forameliorating the effects of exposure to the toxin.

Embodiment 25 comprises use of the IP-MSC of any one of embodiments 1 to20 for the manufacture of a pharmaceutical composition for treating aninfection caused by the pathogen or for ameliorating the effects ofexposure to the toxin.

Embodiment 26 comprises use of the IP-MSC of any one of embodiments 1 to20 for the manufacture of a pharmaceutical composition for preventing aninfection caused by the pathogen or preventing toxicity from exposure tothe toxin.

Embodiment 27 comprises a method for treating an infection caused by thepathogen or treating exposure to the toxin comprising administering to asubject a therapeutically effective dosage of the IP-MSC of any one ofembodiments 1 to 20.

Embodiment 28 comprises a method for preventing an infection caused by apathogen or preventing toxicity from exposure to the toxin comprisingadministering to a subject a prophylactic dosage of the IP-MSC of anyone of embodiments 1 to 20.

Embodiment 29 comprises a method for treating or preventing outbreak ofa disease caused by a pathogen or ameliorating exposure to a toxincomprising the step of administering immunoprotective primarymesenchymal stem cells (IP-MSC) to a subject exposed to or at risk ofbeing exposed to the pathogen or toxin; wherein the IP-MSC aretransfected with one or more episomal vectors encoding at least twoimmunoreactive polypeptides, which specifically target the pathogen ortoxin, each immunoreactive polypeptide comprising an antigen-bindingregion of a neutralizing antibody specific for the pathogen or toxin, orencoding a variant of the antigen-binding region, wherein the variantincludes one or more substitutions in the amino acid sequence of theantigen-binding region and shares at least 50% sequence identity withthe antigen-binding region of the neutralizing antibody.

Embodiment 30 comprises the method of embodiment 29 including theadditional step of transfecting primary mesenchymal stem cells with oneor more episomal vectors encoding the at least two immunoreactivepolypeptides.

Embodiment 31 comprises the method of embodiment 30 including theadditional step of identifying neutralizing antibodies to the pathogenor toxin from one or more blood samples obtained from one or moresurvivors of the pathogenic disease or toxin exposure, prior totransfecting the primary MSC.

Embodiment 32 comprises the method of any one of embodiments 29 to 31including the additional step of preparing one or more episomal vectorsencoding expressible amino acid sequences of the at least two or moreimmunoreactive polypeptides prior to transfecting the primary MSC.

Embodiment 33 comprises the method of any one of embodiments 29 to 32wherein the IP-MSC are selected from any one of embodiments 1 to 20.

Embodiment 34 comprises a method for preparing prophylactic ortherapeutic mesenchymal stem cells for treating or preventing anoutbreak of a disease caused by a pathogen or for ameliorating exposureto a toxin comprising the step of transfecting primary mesenchymal stemcells with one or more episomal vectors encoding at least twoimmunoreactive polypeptides comprising an amino acid from anantigen-binding region of a neutralizing antibody specific for thepathogen or toxin, or encoding a variant of the antigen-binding regionsequence, to produce immunoprotective primary mesenchymal stem cells(IP-MSC) that express the immunoreactive polypeptides; wherein thevariant includes one or more substitutions in the amino acid sequencefrom the antigen-binding region and shares at least 50% sequenceidentity with the antigen-binding region sequence of the neutralizingantibody.

Embodiment 35 comprises the method of embodiment 34 including theadditional step of identifying neutralizing antibodies to the pathogenor toxin from a blood sample obtained from one or more survivors of thepathogenic disease or toxin exposure prior to transfecting the primaryMSC.

Embodiment 36 comprises the method of embodiment 34 or embodiment 35including the additional step of preparing one or more episomal vectorsencoding expressible amino acid sequences of the at least two or moreimmunoreactive polypeptides prior to transfecting the primary MSC.

Embodiment 37 comprises the method of any one of embodiments 34 to 36wherein the IP-MSC are selected from any one of embodiments 1 to 20.

Embodiment 38 comprises an episomal vector encoding an expressible aminoacid sequence of an immunoprotective polypeptide; wherein eachimmunoreactive polypeptide comprises an amino acid sequence from anantigen-binding region of a neutralizing antibody specific for anantigen produced by the pathogen or specific for the toxin, or an aminoacid sequence of a variant of the antigen-binding region sequencecomprising one or more substitutions in the amino acid sequence thereofand sharing at least about 50% sequence identity with theantigen-binding region sequence; the antigen-binding region sequence orvariant thereof being arranged to specifically bind to and neutralizethe pathogen or toxin; and wherein the episomal vector optionallyincludes an inducible apoptosis gene.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 provides a schematic illustration of a full-length IgG antibody,a ScFV, a tandem diabody, and a tribody.

FIG. 2 schematically illustrates an episomal vector for transfectingprimary MSC as described herein (Panel A); and a bicistronic vector usedto transfect human adipose MSC to express human anti-LASV MAb GP19.7E(Panel B).

FIG. 3 illustrates nucleotide sequences of the heavy chains of humananti-LASV IgG MAb GP10.4B (SEQ ID NO: 1) and human anti-LASV MAb GP19.7E(SEQ ID NO: 1).

FIG. 4 illustrates nucleotide sequences of the light chains of humananti-LASV IgG MAb GP10.4B (SEQ ID NO: 3) and human anti-LASV MAb GP19.7E(SEQ ID NO: 4).

FIG. 5 illustrates amino acid sequences of the heavy and light chains ofhuman anti-LASV IgG MAb GP10.4B (HC: SEQ ID NO: 5; LC: SEQ ID NO: 7) andhuman anti-LASV MAb GP19.7E (HC: SEQ ID NO: 6; LC: SEQ ID NO: 8).

FIG. 6 provides a graph of percentage of survivors versus day post LASVinfection for guinea pigs treated with human anti-LASV IgG MAb GP10.4Band human anti-LASV MAb GP19.7E, compared to control guinea pigs treatedwith antibody-free medium.

DETAILED DESCRIPTION OF SELECTED EMBODIMENTS

Immunoprotective primary mesenchymal stems cells described hereinepisomally express multiple immunoreactive polypeptides thatspecifically target a pathogen or toxin of interest. Each immunoreactivepolypeptide comprises an amino acid sequence of an antigen-bindingregion of a neutralizing antibody specific for an antigen produced bythe pathogen or specific for the toxin, or an amino acid sequence of avariant of the antigen-binding region comprising one or moresubstitutions in the amino acid sequence thereof and sharing at leastabout 50% sequence identity with the antigen-binding region. Theantigen-binding region or variant thereof is arranged and oriented tospecifically bind to and neutralize the pathogen or toxin, i.e., whenthe IP-MSC are contacted with the pathogen or toxin, for example whenthe IP-MSC are administered to a subject and the subject is exposed tothe pathogen or toxin. The substitutions in the variant can comprise orconsist of conservative substitutions, or in some cases non-conservativesubstitutions that enhance the binding affinity or binding selectivityof the variant relative to the native antigen-binding region, or whichimprove, enhance or otherwise desirably affect one or more properties ofthe immunoreactive polypeptides, such as a physical, chemical, orconformational property.

The IP-MSC potentially can be utilized against any pathogen or toxin forwhich neutralizing antibodies can be identified. The IP-MSC and methodsdescribed herein are particularly useful for providing a relativelyrapidly deployable, but short-term (e.g., up to one or two months)passive immunity against a pathogen or toxin. Such pathogens includeviruses, bacteria, parasites (single cell and multicellular parasites),and the like. For example, the IP-MSC can be utilized as a protectiveagent in the case of a deliberate or accidental release of a pathogen ortoxin. In addition, IP-MSC against particular pathogenic viruses (e.g.,LASV, Ebola virus, Dengue virus), bacteria (Rickettsia typhi, Neisseriameningitidis, Borrelia spp., Vibrio cholerae, and the like) or parasites(e.g., Plasmodium, Trypanosoma, Leishmania, Schistosoma, and the like)can be utilized as a temporary protection for subjects traveling toareas where the pathogens are endemic, or for prevention of infectionscommonly acquired by patients in hospitals (e.g., Methicillin-ResistantStaphylococcus Aureus, Psuedomonas Aeruginosa, Vancomycin-ResistantEnterococci, Streptococcus pneumoniae, and the like).

An important contributing factor to therapeutics designed around MSC isthe ease of MSC isolation and expansion in culture. Theoretically, asingle bone marrow harvest of MSC may yield sufficient MSC for thousandsof clinical applications, due to their inherent expansion capability(Newman et al., 2009). Such expansion potential greatly enhances the GMPmanufacturing capability of using MSC for clinical applications and haslower production costs when compared to other cell types.

As used herein, the term “immunoreactive polypeptide” and grammaticalvariations thereof refers to a polypeptide that includes a peptideencoding an antigen-binding region of a neutralizing antibody to thepathogen or toxin of interest, or a variant of the antigen-bindingregion which retains specificity for the pathogen or toxin, but differsfrom a native antibody structure by the presence of one or moresubstitution (e.g., a conservative substitution) in the amino acidsequence of the native antigen-binding region. Non-limiting examples ofimmunoreactive polypeptides include full length antibodies (e.g., an IgGantibody), antigen-binding fragments of such full length antibodies, andother polypeptides that include one or more complementarity determiningregion (CDR) of such antibodies arranged and oriented to bind to anantigen. Functional antigen-binding antibody fragments include Fab,F(ab′)₂, Fv, ScFv, diabody, and tribody polypeptides.

As used herein, the term “antigen-binding region” refers to the site ofan antibody that binds to an antigen. The antigen-binding region iscomprised of heavy chain and light chain variable domains (V_(H) andV_(L)), each of which includes four conserved framework regions (FR) andthree CDRs. The CDRs vary in sequence and determine the specificity ofthe antibody to a particular antigen. The V_(H) and V_(L) domainstogether form the site that specifically binds a particular antigen.

Fab (fragment antigen binding) antibody fragments are immunoreactivepolypeptides comprising monovalent antigen-binding domains of anantibody composed of a polypeptide consisting of a heavy chain variableregion (V_(H)) and heavy chain constant region 1 (C_(H)1) portion and apoly peptide consisting of a light chain variable (V_(L)) and lightchain constant (C_(L)) portion, in which the C_(L) and C_(H)1 portionsare bound together, preferably by a disulfide bond between Cys residues.

A F_(V) antibody fragment is a dimer that contains the V_(H) and V_(L)domains.

A F(ab′)₂ fragment is composed of two Fab-type polypeptides boundtogether by a disulfide bridge between the C_(H)1 portions thereof.

A ScFV (“single chain fragment variable” or “single chain antibody”) isan immunoreactive polypeptide comprising V_(L) and V_(H) peptides joinedtogether by a flexible, generally hydrophilic linking peptide, ofsufficient length (generally about 15 amino acids in length) to allowthe V_(L) and V_(H) to associate in an antigen-binding configuration.One common flexible linking peptide is (Gly₄Ser)₃. Optionally, theassociation of the V_(H) and V_(L) can be stabilized by one or moreintermolecular disulfide bonds.

As used herein and as commonly understood in the art, the term “diabody”refers to an immunoreactive polypeptide comprising either (a) two ScFVlinked together by a short peptide or bond between two ScFV (e.g.,between the V_(L) portions) to form a tandem dimeric ScFV or (b) acomplex comprising two ScFV-like polypeptides in which the linkingpeptide is too short to allow direct interaction between the V_(L) andV_(H) of the same polypeptide chain so that two such molecules areforced to associate intermolecularly as a dimer. The two antigen-bindingdomains of the diabody can be specific for the same antigen or twodifferent antigens.

As used herein and as commonly understood in the art, the term “tribody”refers to an immunoreactive polypeptide comprising three ScFV-likeantigen binding domains. Structurally, a tribody is a dimer composed oftwo polypeptide chains bound together by a disulfide bridge, in whichthe first polypeptide comprises an ScFV linked to an additional V_(L)domain through a C_(L) polypeptide chain, and the second polypeptidecomprises an ScFV linked to an additional V_(H) domain through a C_(H)1polypeptide chain. The disulfide bridge is formed between a Cys residuein the C_(L) and a Cys residue in the C_(H)1, such that the additionalV_(L) of the first polypeptide associates with the additional V_(H) ofthe second polypeptide in an antigen-binding configuration, such thatthe tribody as a whole includes three antigen-binding domains. The threeantigen-binding domains of the tribody can be specific for the sameantigen or two or three different antigens.

FIG. 1 schematically illustrates a full length IgG antibody, an ScFV, atandem-type diabody, and a tribody as discussed above.

The use of the terms “a” and “an” and “the” and similar referents in thecontext of describing the invention (especially in the context of thefollowing claims) are to be construed to cover both the singular and theplural, unless otherwise indicated herein or clearly contradicted bycontext. The terms “comprising,” “having,” “including,” and “containing”are to be construed as open-ended terms (i.e., meaning “including, butnot limited to,”) unless otherwise noted. Recitation of ranges of valuesherein are merely intended to serve as a shorthand method of referringindividually to each separate value falling within the range, unlessotherwise indicated herein, and each separate value is incorporated intothe specification as if it were individually recited herein. All methodsdescribed herein can be performed in any suitable order unless otherwiseindicated herein or otherwise clearly contradicted by context. The useof any and all examples, or exemplary language (e.g., “such as”)provided herein, is intended merely to better illuminate the inventionand does not pose a limitation on the scope of the invention unlessotherwise claimed. No language in the specification should be construedas indicating any non-claimed element as essential to the practice ofthe invention.

Preferably, the immunoprotective polypeptides share at least about 50%sequence identity with the antigen-binding region of a naturallyoccurring (native) antibody (e.g., at least about 55, 60, 65, 70, 75,80, 85, 90, 95, 98, or 99% sequence identity with the naturallyoccurring antibody antigen-binding region). As used herein, the terms“naturally occurring antibody”, “native antibody” and grammaticalvariations thereof refer to an antibody specific for the pathogen ortoxin of interest, which is identified from a blood sample of a subjectexposed to the pathogen or toxin.

Non-limiting examples of viral pathogens that can be targeted by theimmunoprotective polypeptides produced by the IP-MSC described hereininclude: adenoviruses; papillomaviruses; hepadnaviruses (e.g., hepatitisB); parvoviruses; pox viruses (e.g., small pox virus, vaccinia virus);Epstein-Barr virus; cytomegalovirus (CMV); herpes simplex viruses;roseolovirus; varicella zoster virus; filoviruses (e.g., Ebola virus andMarburg virus); paramyxoviruses (e.g., measles virus, mumps virus, Nipahvirus, Hendra virus, human respiratory syncytial virus (RSV),parainfluenza viruses, Newcastle disease virus, and humanmetapneumovirus); orthomyxoviruses (e.g., influenza A, influenza B, andinfluenza C); rhabdoviruses (e.g., Lyssavirus, also known as rabiesvirus); arenaviruses (e.g., Lassa virus); coronaviruses (severe acuterespiratory syndrome (SARS)); human enteroviruses; hepatitis A virus;human rhinoviruses; polio virus; retroviruses (e.g., humanimmunodeficiency virus 1(HIV-1)); rotaviruses; flaviviruses, (e.g., WestNile virus, dengue virus, yellow fever virus); hepaciviruses (e.g.,hepatitis C virus); and rubella virus.

Non-limiting examples of bacterial pathogens that can be targeted by theimmunoprotective polypeptides produced by the IP-MSC described hereininclude any pathogenic bacterial species from a genus selected from:Bacillus; Bordetella; Borrelia; Brucella; Burkholderia; Campylobacter;Chlamydia, Chlamydophila; Clostridium; Corynebacterium; Enterococcus;Escherichia; Francisella; Haemophilus; Helicobacter; Legionella;Leptospira; Listeria; Mycobacterium; Mycoplasma; Neisseria; Pseudomonas;Rickettsia; Salmonella; Shigella; Staphylococcus; Streptococcus;Treponema; Vibrio; and Yersinia.

Non-limiting examples of parasitic pathogens that can be targeted by theimmunoprotective polypeptides produced by the IP-MSC described hereininclude single cell and multicellular parasites, such as: Acanthamoeba;Anisakis; Ascaris lumbricoides; Balantidium coli; Cestoda (tapeworm);Chiggers; Cochliomyia hominivorax; Entamoeba histolytica; Fasciolahepatica; Giardia lamblia; Hookworm; Leishmania; Linguatula serrata;Liver fluke; Loa loa; Paragonimus (lung fluke); Pinworm; Plasmodiumfalciparum; Schistosoma; Strongyloides stercoralis, Tapeworm, Toxoplasmagondii; Trypanosoma; Whipworm; and Wuchereria bancrofti.

Non-limiting examples of viral antigens that can be targeted by theimmunoprotective polypeptides produced by the IP-MSC described hereininclude: influenza polypeptides such as hemagglutinin 1 (HA1),hemagglutinin 2 (HA2), and neuraminidase (NA); Lassa virus (LASV)polypeptides such as LASV glycoprotein 1 (gp1), LASV glycoprotein 2(gp2), LASV nucleocapsid-associated protein (NP), LASV L protein, andLASV Z protein; SARS virus polypeptides such as SARS virus S protein;Ebola virus polypeptides such as Ebola virus GP2; measles viruspolypeptides such as measles virus fusion 1 (F1) protein; HIV-1polypeptides such as HIV transmembrane (TM) protein, HIV glycoprotein 41(gp41), HIV glycoprotein 120 (gp120); hepatitis C virus (HCV)polypeptides such as HCV envelope glycoprotein 1 (E1), HCV envelopeglycoprotein 2 (E2), HCV nucleocapsid protein (p22); West Nile virus(WNV) polypeptides such as WNV envelope glycoprotein (E); Japaneseencephalitis virus (JEV) polypeptides such as JEV envelope glycoprotein(E); yellow fever virus (YFV) polypeptides such as YFV envelopeglycoprotein (E); tick-borne encephalitis virus (TBEV) polypeptides suchas TBEV envelope glycoprotein (E); hepatitis G virus (HGV) polypeptidessuch as HGV envelope glycoprotein 1 (E1); respiratory synctival virus(RSV) polypeptides such as RSV fusion (F) protein; herpes simplex virus(HSV) polypeptides such as HSV-1 gD protein, HSV-1 gG protein, HSV-2 gDprotein, and HSV-2 gG protein; hepatitis B virus (HBV) polypeptides suchas HBV core protein; and Epstein-Barr virus (EBV) polypeptides such asEBV glycoprotein 125 (gp125).

Non-limiting examples of bacterial antigens that can be targeted by theimmunoprotective polypeptides produced by the IP-MSC described hereininclude: outer membrane protein assembly factor BamA; translocationassembly module protein TamA; polypeptide-transport associated proteindomain protein; bacterial surface antigen D15 from a wide variety ofbacterial species; Bacillus anthracis polypeptides such as anthraxprotective protein, anthrax lethal factor, and anthrax edema factor;Salmonella typhii polypeptides such as S1Da and S1Db; Vibrio choleraepolypeptides such as cholera toxin and cholera heat shock protein;Clostridium botulinum polypeptides such as antigen S and botulinumtoxin; and Yersina pestis polypeptides such as F1, V antigen, YopH,YopM, YopD, and plasminogen activation factor (Pla).

Non-limiting examples of parasite antigens that can be targeted by theimmunoprotective polypeptides produced by the IP-MSC described hereininclude: malarial (Plasmodium) polypeptides such as circumsporozoiteprotein (CSP), sporozoite surface protein (SSP2/TRAP), liver stageantigen 1 (LSA1), exported protein 1 (EXP 1), erythrocyte bindingantigen 175 (EBA-175), cysteine-rich protective antigen (cyRPA), andPlasmodium heat shock protein 70 (hsp70); and Schistosoma polypeptidessuch as Sm29 and signal transduction protein 14-3-3.

Preferably, the IP-MSC are administered parenterally (e.g. intravenous,subcutaneous, or intramuscular injection or infusion). The IP-MSC can beformulated as a solution, suspension, or emulsion in association with apharmaceutically acceptable carrier vehicle (e.g., sterile water,saline, dextrose solution, phosphate buffered saline, and similarmaterials suitable for administration of live stem cells). Optionally,additives that maintain isotonicity (e.g. mannitol) or chemicalstability (e.g. preservatives) can be included in the carrier.

As used herein, a “therapeutically effective dosage” is an amount (e.g.,number of IP-MSC) such that when administered, the IP-MSC result in areduction or elimination of already present disease symptoms (e.g.,about one hundred thousand to about one hundred million cells). Thedosage and number of doses (e.g. single or multiple dose) administeredto a subject will vary depending upon a variety of factors, includingthe route of administration, patient conditions and characteristics(sex, age, body weight, health, size), extent of symptoms, concurrenttreatments, frequency of treatment and the effect desired, the identityand number of antigenic polypeptides expressed by the IP-MSC, and thelike. Adjustment and manipulation of established dosage ranges, as wellas in vitro and in vivo methods of determining the therapeuticeffectiveness of the IP-MSC in an individual, are well within theability of those of ordinary skill in the medical arts.

A “prophylactic dosage” is an amount (e.g., number of IP-MSC) such thatwhen administered, the MSC prevent infection by the pathogen from whichthe polypeptide expressed by the IP-MSC was derived (e.g., about onehundred thousand to about one hundred million cells). The dosage andnumber of doses (e.g. single or multiple dose) administered to a subjectwill vary depending upon a variety of factors, including the route ofadministration, patient conditions and characteristics (sex, age, bodyweight, health, size), extent of symptoms, concurrent treatments,frequency of treatment and the effect desired, the identity and numberof antigenic polypeptides expressed by the IP-MSC, and the like.Adjustment and manipulation of established dosage ranges, as well as invitro and in vivo methods of determining the prophylactic effectivenessof the IP-MSC in an individual, are well within the ability of those ofordinary skill in the medical arts.

As used herein, the term “episomally transfected” and grammaticalvariations thereof refer to non-integrating transfection with exogenousepisomal DNA (e.g. a plasmid or other episomal vector) to produce a cellwith unaltered chromosomal DNA, in which the polypeptide encoded by theDNA is expressed in an episome within the MSC, i.e., without genomicintegration of the exogenous DNA. As used herein, the term “episome” andgrammatical variations thereof refers to closed circular DNA moleculesthat are replicated in the nucleus, and is intended to encompassexogenous plasmids introduced into the MSC. Preferably, primary MSC aretransfected with a plasmid that encodes the antigenic polypeptide, andpreferably also encodes regulatory elements (e.g., a promoter) tofacilitate episomal expression of the antigenic polypeptide. Optionally,the also MSC can be episomally transfected with an inducible apoptosisgene to induce cell death (apoptosis) when activated by a suitablesignal (e.g., using Tetracycline-Controlled Transcriptional Activation,also referred to as “Tet-on and Tet-off”, in which tetracycline ordoxycycline is used to turn on transcription of the apoptotic gene), sothat the IP-MSC can be eliminated from the subject if desired or needed(e.g., if undesired side-affects develop). The term “episomal vector”refers to an expression vector comprising a plasmid or other circularDNA encoding the antigenic polypeptide.

Primary MSC can be episomally transfected by any suitable methodology.For example, the Primary MSC can be transfected with a plasmid encodingthe antigenic polypeptide using electroporation, lipofection, and thelike. Electroporation is the preferred method for transfection, unlikeother transfection approaches using cationic lipids (i.e. lipofection)as there may be residual lipids after transfection that may not becompletely removed when processing the MSC for delivery, and may resultin unforeseen side effects.

Non limiting examples of episomal vectors suitable for use asnon-integrating vectors for transfection of eukaryotic cells (e.g.,primary MSC) include simian virus 40-based vectors, Epstein-Barrvirus-based vectors, papilloma virus-based vectors, BK virus-basedvectors, and the like, which are well known in the molecular geneticsart.

Also described herein is a method for treating or preventing apathogenic disease or ameliorating exposure to a toxin, utilizing theIP-MSC described herein. One method embodiment comprises the steps of:optionally identifying neutralizing antibodies to the pathogen or toxinidentified from a blood sample from one or more survivors of thepathogenic disease or toxin exposure; transfecting primary mesenchymalstem cells with one or more episomal vectors encoding at least twoimmunoreactive polypeptides comprising an antigen-binding region of aneutralizing antibody specific for the pathogen or toxin (e.g., anantibody identified in step (a)), or encoding a variant of theantigen-binding region, to produce IP-MSC that express theimmunoreactive polypeptides; and administering the IP-MSC to a subjectexposed to or at risk of being exposed to the pathogen or toxin. Thevariant, if utilized, includes one or more substitutions in the aminoacid sequence of the antigen-binding region preferably shares at least50% sequence identity with the antigen-binding region of theneutralizing antibody.

Selection and Design of Antibodies and Immune Molecules.

Advanced methods for transport of immune cells from survivors ofexposure to pathogenic agents or toxins. Isolation and cryopreservationof peripheral blood mononuclear cells (PBMC) from blood samples shouldpreserve the integrity of as many B cells as is practical, to ensureeventual recovery and characterization of abundant and rarespecificities. As proof of concept, convalescent Lassa fever (LF)patients at West African clinical research sites (Kenema GovernmentHospital, Sierra Leone and Irrua Specialist Teaching Hospital, Nigeria)are identified using modern recombinant protein-based immunodiagnostics.Whole blood is drawn after fully informed consent, and PBMC are isolatedin dedicated and fully equipped cell culture suites. PBMC arecryopreserved using established buffers (RPMI/20% FBSΔ/10% DMSO) andmethods (cooling rate of about −1° C./min to a final temperature ofabout −80° C., >5×10⁶ cells/vial) that generate highly viable cellcultures after thawing. Samples are rapidly transported to the U.S. inIATA-approved cryogenic containers for further processing. The numberand percentage of B cells prior to cryopreservation are assessed onsiteby quantitative flow cytometry (with the aid of a highly portable BDACCURIC6 cytometer), by determining total number of PBMC, andspecifically B cells (CD19+, CD20+), T cells (CD3+, CD 4+, CD 8+), NKcells (CD16+, CD 56+), and monocytes (CD14+, CD 16+) from each isolationprocedure. The procedure is repeated upon thawing to determine lossrates of PBMC and cell subsets. Similar procedures can be employed inidentifying other antibodies, e.g., influenza antibodies, produced byPBMC from subjects with documented recent infections. Where appropriate,as in influenza, cryopreservation might be bypassed, permittingisolation of B cells from fresh blood draws.

Methodology for rapid determination of the microbiome in an indexpatient convalescing from a pathogen or toxin. To demonstrate that theMSC gene delivery platform can be deployed rapidly as a firebreak for ahigh-risk group (warfighters, first responders, etc.) against a highlytransmissible disease a novel pathogen identified in Sierra Leone orNigeria is used as a model. This closely replicates or simulates thescenario of a patient accessible about 2 to 3 weeks following exposureto a pathogen or toxin (e.g., of known or unknown origin), as well asdeliberately-released or accidentally-released pathogens or toxins.

Microbial metagenomics, the unbiased characterization of microbialnucleic acids, can rapidly identify infectious pathogens in patientsconvalescing from unknown biothreats. Microbes present in clinicalsamples are typically identified by culture or by targeted molecularapproaches, such as PCR or antigen capture. Culturing is time-consumingand many microbes simply cannot be cultured in vitro. Targetedapproaches are also disadvantageous because they require a prioriknowledge of the organism. About 30% of microbial reads in fever ofunknown origin (FUO) samples from Sierra Leone and Nigeria have no matchin the GENBANK database. The technology described herein is sensitive(i.e., able to detect a low-copy pathogen in a diverse mixture ofendogenous microbes) and scalable (i.e., able to survey large numbers ofpatient samples quickly). Improved molecular methods for constructingIllumina sequencing libraries are developed using sub-nanogramquantities of RNA. Current library construction methods are scaled up sothat hundreds of samples can be processed in parallel. A bioinformaticspipeline is developed that can rapidly identify all microbes present inmassive next-generation sequencing data sets. With these methods inplace, a complete genome is assembled from an unknown virus within about2 days and an unknown bacterium within about 4 days.

Computer programs for identifying pathogen virulence determinants (e.g.viral entry glycoproteins or toxins). Virulence factors refer to theproteins (i.e., gene products) that enable a microorganism to establishitself in humans and enhance its potential to cause disease. Abioinformatics and computational pipeline that can rapidly identifyvirulence factors of previously unknown organisms from large metagenomicdatasets is developed that leverages several publicly availablevirulence factor databases (MvirDB, Tox-Prot, SCORPION, the PRINTSvirulence factors, VFDB, TVFac, Islander, ARGO and a subset of VIDA) andenables rapid (within a few hours) identification of virulence factorsfor newly completed genomic sequence data. Experiments are performed toconfirm the ability of the program to correctly identify virulencefactors that can be targeted for protective antibodies.

Protective antibodies. B cells are enriched by depletion of non-B cellsfrom PBMC using a MACS separator (Miltenyi Biotec) and magnetic beadscoated with, e.g., αCD2, CD4, CD11b, CD16, CD36, αIgE, CD235a, and thelike. Antigen-specific circulating memory B cells that bind tofluorochrome-labeled recombinant virulence determinants (e.g. LASV GP orinfluenza virus hemagglutinin, HA) are sorted by flow cytometry anddirectly deposited in 96 well plates at single cell densities. RNA isisolated from single cells (Norgen Biotek, Qiagen) and reversetranscribed into cDNA, with subsequent amplification of immunoglobulinheavy and light chains. Custom designed oligos permit direct cloning ofheavy and light chain genes into proprietary vectors for expression ascomplete human monoclonal antibodies (CHOLCelect™, U.S. Pat. No.8,076,102, Luis M Branco et al., which is incorporated herein byreference in its entirety), or re-engineered as single chain, diabodies,or tribodies for rapid expression and purification from E. colicultures. ELISAs for binding to virulence determinants (GP or HA) orpathogen neutralization (pseudoparticle-based) assays are used torapidly identify potentially protective antibodies. Antibodies withdesired properties are cloned in the polyclonal expression vectordescribed herein, and are tested as single specificities in live virusneutralization assays to verify potency in a more relevant in vitrobiological system.

Methods for avoiding formation of chimeric improperly assortedantibodies. To ensure the development of the most widely applicable,protective, and escape mutant-resistant platform an oligoclonal antibodydesign is implemented. To this end each antibody displaying neutralizingproperties desirably is tested as full length and single chain (ScFV)versions. Many ScFV preserve the properties of parent whole IgGs due tofaithful representation of crucial antigen-binding CDRs. Alternatively,diabody and tribody combinations can be incorporated in expressionvector platforms for therapeutic use. This IgG/ScFV oligoclonal approachwill prevent formation of inappropriately mixed heavy and light chainswhen more than one antibody is expressed within a single MSC. In someembodiments, a desired level of therapeutic potency is achieved byintegrating several ScFVs and one full length antibody for whicheffector functions may be identified or that lost potency uponconversion to a single chain variant.

Selection and Design of Nucleic Acid Constructs.

Vector design to allow for sufficient levels and duration of expressionfor protection against challenge. A multicopy, non-infective,non-integrative, circular episome is used to express protectivecompletely human single chain antibody fragments, full length IgGs, orother immunoreactive polypeptides against multiple (potentiallyhundreds) bacterial, viral, fungal, or parasite proteins or proteintoxoids simultaneously (see FIG. 1, which illustrates IgG, ScFV, diabodyand tribody-type immunomodulators). In some preferred embodiments, theepisome is based on components derived from Epstein-Barr virus (EBV)nuclear antigen 1 expression cassette (EBNA1) and the OriP origin ofreplication. These preferably are the only components of EBV that areused, so that no viruses are replicated or assembled. This systemresults in stable extra-chromosomal persistence and long-term ectopicgene expression in mesenchymal stem cells. In the methods describedherein, ScFVs or other immunoreactive polypeptides are effectivelyexpressed in and secreted from MSC in protective amounts. A full lengthLASV neutralizing antibody in hADMSC has been expressed as a proof ofconcept. The ability of EBV-based episomes to introduce and maintainvery large human genomic DNA fragments (>300 kb) in human cells isanother significant advantage of the methods described herein. Thisfeature permits cloning of dozens of expression elements in a vectorcapable of replicating in bacteria, amenable to large scalepurification, transfection into hMSC, and replication as an episomalplasmid. Targeted expression levels for the immunoreactive polypeptides(e.g., ScFVs) are about 10 pg/cell/day for each immunoreactivepolypeptide, preferably expression levels of 5 pg/cell/day. An infusionwith about 1×10¹¹ MSC with a productivity rate of 10 pg/cell/day foreach immunoreactive polypeptide generates about 1 gram of solublepolypeptide per day, equivalent to a 15 mg/mL level in the circulationof a 75 Kg adult, which is a suitable therapeutic dosage level.Promoters and other regulatory elements are used to drive the expressionof each type of immunomodulatory molecule.

Several reports in the literature point to a non-classical pattern ofexpression from well characterized promoters in MSC. The humancytomegalovirus major immediate early gene promoter (CMV-MIE) is one ofthe strongest promoters known, and a major element in the generation ofmulti-gram per liter recombinant protein drug producing stable mammaliancell lines. The CMV-MIE is however, relatively poorly transcribed inMSC. In contrast, EF1A, UBC, and CAGG promoters have demonstrated highlevels of expression in MSC without obvious signs or promoter silencing.The episomal vectors utilized in the methods described herein caninclude any such promoters. FIG. 2, Panel A, provides a schematicillustration of a representative and non-limiting example of a pEBV MSCepisomal vector. Expression vectors without antibiotic selection markersalso are provided for expansion of plasmids in E. coli. The replicativenature of the episomal plasmid precludes its linearization with arestriction endonuclease that disrupts the antibiotic resistance gene'sopen reading frame. Thus, it is conceivable that genetic rearrangementswould result in expression of an antibiotic resistance gene, potentiallygiving rise to undesirable antibiotic resistance-mediated side effectsin humans in selected cases. This scenario can be averted bysubstituting antibiotic resistance genes with metabolic selectablemarkers for growth and propagation of plasmids in E. coli strains, ifneeded or desired.

Design of regulatory elements for targeted expression levels ofindividual therapeutic molecules and shutoff. Regulatory elements in thevector are utilized to accommodate desired secreted levels and serumlevels of each immunomodulatory molecule of interest. Expression of fulllength antibodies, ScFV, or other immunoreactive polypeptides benefitfrom strong promoters (e.g. CMV, EF1A, CAGG, etc.) to achievetherapeutic serum levels within less than one day after administrationof MSCs. Other immunomodulatory molecules, such as cytokines, are oftenexpressed and secreted at low levels, and transiently by MSC. Toaccommodate required flexibility in disparate levels and timing ofexpression such genes are driven from low basal promoters (i.e. TK), orthrough controlled induction from a Tet on/off promoter. The Tetpromoter system benefits from the use of innocuous antibiotic analogssuch as anhydrotetracycline, which activates the Tet promoter atconcentrations 2 logs lower than with tetracycline, does not result indysregulation of intestinal flora, does not result in resistance topolyketide antibiotics, and does not exhibit antibiotic activity.Anhydrotetracycline is fully soluble in water, and can be administeredin drinking rations to potentiate activation of selected genes intransfected MSCs. The potential toxicity of anhydrotetracycline, thefirst breakdown product of tetracycline in the human body, can becircumvented by administration of other analogs, such doxycycline, anFDA-approved tetracycline analog that also activates the Tet on/offpromoter system. This system preferentially is employed in the design ofa failsafe “kill switch” by tightly regulating inducible expression of apotent pro-apoptotic gene (e.g. Bax) to initiate targeted apoptosis oftransfected MSCs in the event of untoward side effects or when thedesired therapeutic endpoint has been achieved. Recent advances in theTet-on system have resulted in much enhanced repression of promoterleakiness and responsiveness to Dox at concentrations up to 100-foldlower than in the original Tet system (Tet-On Advanced™, Tet-On 3G™).Drug selectable markers are not used to maintain vector stability intransfected MSC: EBV-based vectors, which are known to replicate and beretained in daughter cells at a rate of 90-92% per cell cycle.

Vector safety/immunogenicity studies. Because episomes do not producereplicating viruses, and the cells in which they are expressed do notproduce MHC molecules in any significant amounts, episomes do not resultin vector-derived immunity that would prevent a subsequent use of theplatform in an individual. This can be confirmed by designing asensitive assay to detect immune responses (antibody ELISA and T-cellbased assays) to components derived from Epstein-Barr virus (EBV)nuclear antigen 1 expression cassette, and to the MSC background (HLAtyping). Genetic studies are performed to investigate rates of EBVintegration into the host cell chromosome (FISH, Southern blot, qPCR),and to measure the transient replicative nature of the vector. It hasbeen reported that EBV vectors retain about 90 to 92% replication percell cycle in the absence of a selectable marker. A decreasingreplication rate contributes to the clearance of the vector from thehost system. Compartmentalization of injected MSC is assessed innon-human primates (NHP) by tracking fluorescently labeled cellspreloaded with cell membrane permeable dyes (green CMFDA, orange CMTMR)that upon esterification will no longer cross the lipid bilayer andbecome highly fluorescent. Such measurements are performed on freshlyprepared tissue sections (lymph nodes, liver, spleen, muscle, brain,pancreas, kidney, intestine, heart, lung, eye, male and femalereproductive tissue) or through whole body scans. Additional tissuesections are processed for isolation of DNA and RNA for analysis ofvector sequences and corresponding transcripts. Design of oligosspecific for each immunoreactive polypeptide, cytokine, and shutofftranscript permit assessment of individual gene expression in alltissues. Some promoters are more actively transcribed in some tissuesthan others, requiring assessment of both the preferential localizationof MSC to peripheral tissues after injection and MSC residency and thecorresponding transcriptional activity of the recombinant genes. To thisend, two artificial “barcode” nucleic acids tags can be included, onespecific to Tet on/off-driven RNA transcripts, and the other to episomalvector DNA. These tags permit rapid identification of the very uniquesequences among the NHP and human genome and transcriptome background(see FIG. 2).

Selection and Design of the Delivery Strategy.

MSC as transient delivery vehicles for therapeutic molecules. MSC areamenable to large scale electroporation, with up to 90% efficiency.MaxCyte, Inc. (Gaithersburg, Md.) markets the “MaxCyte® VLX™ Large ScaleTransfection System, a small-footprint, easy to use instrumentspecifically designed for extremely large volume transient transfectionin a sterile, closed transfection environment. Using flowelectroporation technology, the MAXCYTE VLX can transfect up to about2×10¹¹ cells in less than about 30 minutes with high cell viability andtransfection efficiencies in a sterile, closed transfection environment.This cGMP-compliant system is useful for the rapid production ofrecombinant proteins, from the bench through cGMP pilots and commercialmanufacturing”. MSC can be grown in chemically defined (CD) media, inlarge scale cell culture environments. Recent advances in bioprocessingengineering have resulted in rapid development of CD formulations thatsupport large scale expansion of MSC without loss of pluripotentcharacteristics and retention of genetic stability. Adipose-derived MSCcan be readily procured from liposuction procedures, with an averageprocedure yielding about 1×10⁸ MSC, thus providing sufficient cellnumbers for expansion ex vivo prior to banking (approximately 25doublings, >3×10¹⁵ cells) with remaining lifespan and number ofdoublings (approximately 25) sufficient to sustain expression anddelivery of therapeutic molecules in vivo for several weeks afterinfusion. MSC commonly display doubling rates in the 48 to 72 hourrange, thus potentially providing in vivo lifespans in the range of 50to 75 days. The turnover rate of infused MSC can be assessed bymeasuring circulating levels of transgene products, and by detection ofEBV sequences by qPCR in blood, nasal aspirates, and urine, in humans.Essentially complete elimination of MSC after the desired therapeutictimespan can be achieved by inducing self-destruction via controlledinducible expression of pro-apoptotic genes built into the expressionvector. Levels of circulating MSC-derived immunoreactive polypeptides orother immunomodulators after injection, and vector induced autoimmunityor GVHD responses in NHP also can be assessed. In humans, additionalmarkers associated with autoimmune or allogeneic immune responses can bemeasured, such as biomarkers of liver injury (ALT, AST), liver (ALB,BIL, GGT, ALP, etc.) and renal function markers (BUN, CRE, urea,electrolytes, etc.).

Isolation, characterization, and banking MSC for therapeutic use. Thelack of expression of lymphohematopoietic lineage antigens distinguishesMSCs from hematopoietic cells, endothelial cells, endothelialprogenitors, monocytes, B cells and erythroblasts. Primary MSC are notimmortal and thus are subject to the “Hayflick limit” of about 50divisions for primary cells. Nevertheless, the capacity for expansion isenormous, with one cell capable of producing up to about 10¹⁵ daughtercells. Additionally, MSC have low batch-to-batch variability. Cell banksizes capable of rapidly protecting millions of at risk individuals canbe generated by pooling large numbers of pre-screened donor adiposetissue-derived MSC: 100 donors at 1×10⁸ cells/donor×25 generations exvivo=about 3×10¹⁷ cells; at about 1×10¹¹ cells/infusion=about 3 milliondoses. Two approaches can be used in the generation of therapeutic MSCbanks: (1) isolation, expansion, testing, banking, following bytransfection, recovery and administration; and (2) isolation, expansion,testing, transfection, banking to generate ready-to-administer cellsupon thawing and short recovery.

For characterization, the master cell bank can be tested for sterility,mycoplasma, in vitro and in vivo adventitious agent testing, retrovirustesting, cell identity, electron microscopy, and a number of specificvirus PCR assays (the FDA requires 14 in their 1993 and 1997 guidancedocuments, and that list has been augmented with several recommendedviruses in addition, mainly polyoma viruses). With the potential initialuse of serum in primary culture conditions, testing can be performed forthe 9CFR panel of bovine viruses. If cells come in contact with porcineproducts during normal manipulations testing for porcine virusespreferably is performed, as well.

Pharmacokinetics/pharmacodynamics (PK/PD). One of the limitations ofusing MSC for tissue repair has been the inability of cells topermanently colonize organs after ex vivo expansion and reinjection intothe person from which they were derived. MSC circulate for a limitedperiod of time (e.g., several weeks or months), whether injected intoMHC matched or unmatched individuals. This particular short-coming inthe development of an adult MSC universal gene delivery platform is abenefit in the methods described herein. The pharmacokinetic (PK)profile of each transgene expressed in transfected MSC can be assessedin NHP for each engineered delivery vector platform developed. Onesingle dose PK study desirably is performed in cynomolgus monkeys, withtransfected MSC administered IV. In such a study 2 male and 2 femalemonkeys each are intravenously (i.v.) administered a high dose (about10¹¹ cells), intermediate dose (about 10⁸ cells), and a low dose (about10⁵ cells) of MSC. Endpoints to be evaluated include: cage-sideobservations, body weight, qualitative food consumption, ophthalmology,electrocardiogram, clinical pathology (e.g., hematology, chemistry,coagulation, urinalysis); immunology (e.g., immunoglobulins andperipheral leukocytes such as B cells, T cells and monocytes);immunogenicity; gross pathology (e.g., necropsy and selected organweights); histopathology; tissue binding; and pharmacokinetics. Serumconcentrations of each recombinant antibody can be monitored over 9weeks with qualified sandwich type ELISA that utilize antibody-specificcapture and detection (HRP-labeled anti-id) reagents on days 1, 3, 6,12, 24, 36, 48, and 63. PK analyses can be conducted bynon-compartmental methods using WINNONLIN software (Pharsight Corp.).Pharmacokinetic parameters for each antibody can be expressed as maximumserum concentration (C_(max)), dose normalized serum concentration(C_(max)/D), area under the concentration-time curve from time 0 toinfinity (AUC_(0-∞)), dose normalized area under the concentration-timecurve from time 0 to infinity (AUC_(0-∞)/D), total body clearance (CL),volume of distribution at steady state (V_(ss)), apparent volume ofdistribution during the terminal phase (V_(z)), terminal eliminationphase half-life (t_(1/2,term)), and mean residence time (MRT).Peripheral circulation and compartmentalization of injected MSC can beassessed in NHP by tracking fluorescently labeled cells preloaded withcell membrane permeable CMFDA or CMTMR dyes, as described above, onfreshly prepared tissue sections or through whole body scans. Vector DNAsequences and transcripts can be monitored by qPCR, as outlined above.

Reusability. There is an extensive body of literature outlining the lackof rejection against MSC in vivo. Nonetheless, this phenomenon can beevaluated in NHP with multiple injections of syngeneic MSC modified withhomologous and heterologous DNA vectors, followed by immunologicalprofiling of allogeneic responses. For example, one group of NHP can beinjected with a bolus of syngeneic MSC transfected with an episomalvector expressing LASV antibodies, and another with a similar vectorexpressing influenza antibodies. The immune response to the MSC platformand to components of the vector can be assessed weekly over the courseof 77 days, during which any immunological response should bedetectable. Safety and immunogenicity in NHP following activation of theshutoff mechanism by administration of doxycycline or other tetracyclineanalogs can be assessed in similar fashion. Following administration ofa doxycycline regimen, adverse immunological responses to vectorcomponents and the MSC delivery platform can be assessed in a similarfashion, e.g., first semi-daily for the first 2 weeks, then weekly foran additional 77 days. Additional markers of apoptotic cell death can betracked by established assays, such as increased serum lacticdehydrogenase (LDH) and caspases, and phosphatidyl serine (PS) incirculating MSC. If an immunological response to vector and MSC is notdetectable following this 77-day period NHP can be re-injected withhomologous MSC, one group with MSC transfected with an homologousvector, whereas the other group will receive a heterologous DNA vector.The homologous and heterologous vectors will have the same background,but with different recombinant antibody repertoires. This approach candemonstrate immunogenicity against the MSC and the expression DNAvector, irrespective of the recombinant antibody repertoire. The 77 daytimeline for assessment of immunological reactions against the MSCplatform is chosen based on multiple dose toxicokinetic studies withhuman antibodies in cynomolgus monkeys showing a mean 5000-foldreduction in peak serum levels of recombinant antibody administered at10 mg/Kg over this time frame. In such studies some NHP may developanti-human antibody responses around 50 to 60 days following the firstadministration, while some animals may never develop a detectablehumoral response to the heterologous IgG.

Transport of MSC. Desirably, the MSC can be transported in a device thatallows for warm chain (37° C.) transport of genetically modified MSCallowing for elimination of cold-chain transport, with increased samplecapacity and cell monitoring technologies, such as devices from MicroQTechnologies. These devices maintain precise warm temperatures fromabout 24 to about 168 hours, thereby allowing sufficient time fordeployment of a ready-to-use therapeutic anywhere in the world.Additional capacity for storage and transport of encapsulated cells canbe introduced, and capsules capable of supporting gas exchange can beprepared, as needed. The elapsed time from encapsulation toadministration will account for metabolic changes in IP-MSC, cell growthrate, changes in viability, and any additional product changes that willimpact performance.

Demonstration of Transient Protective Immunity.

Challenge studies in macaques infused with MSC expressing protectiveantibodies. Cynomolgus macaques are infused with macaque MSCs expressinganti-LASV GP single chain antibodies, then challenged by IM injectionwith 1000 plaque forming units (pfu) of LASV virus (Josiah strain), andevaluated as described by Geisbert et al. Animals showing clinical signsconsistent with terminal LF are euthanized. Following challenge,bio-samples are processed for measurement of viremia by plaque assay andRT-PCR. Viral RNA is sequenced to identify whether specific mutations inthe GPC gene occur upon therapy in NHPs. For any animal that succumbs tochallenge, a variety of bio-samples including tissues, blood, and otherbody fluids are taken for histopathology, immunohistochemistry, virusisolation, and genome detection. Surviving macaques are monitored forhumoral responses to viral antigens by conducting a series ofWestern-blot and ELISA assays to detect evidence of any antibodyresponse to major viral structural proteins (G1, G2, NP, Z). Similarstudies with MSC constructs expressing protective antibodies againstinfluenza virus are used for human challenge studies.

The following non-limiting examples are provided to illustrate certainfeatures and aspects of the IP-MSC and methods described herein.

EXAMPLE 1 Lassa Virus (LASV) Neutralizing Antibodies

About thirty milliliters of whole blood were collected from confirmedadult Lassa fever (LF) survivors from Sierra Leone no earlier than 8weeks following discharge from the hospital, and up to several months ofconvalescence. Peripheral blood mononuclear cells (PBMC) were isolatedfrom the blood samples by Ficoll gradient centrifugation, cryopreserved,and transported in dry shippers to the United States. Cultures of thePBMC were plated at low densities in 96-well plates and stimulated withR848 and interleukin-2 (IL-2) for polyclonal activation of B cells.Supernatants from wells showing colony growth after stimulation werescreened for human IgG binding to ELISA plates coated withrecombinantly-expressed LASV NP, GPC (GP1+GP2), GP1, or Z proteins.Clones with significant reactivity were expanded, cloned, andre-screened. RNA was isolated from B cell clones producing IgG specificto LASV proteins. Human light chain (LC) and heavy chain (HC) genes fromthe IgG were amplified by RT-PCR, and cloned in linear single chainexpression vectors. HEK-293T cells were co-transfected with matched LCand HC constructs to assess expression of individual LASV humanmonoclonal antibodies (huMAbs) and to purify small quantities ofantibody for preliminary in vitro characterization studies.

Frozen PBMCs shipped from Sierra Leone had excellent viability and highfrequencies of antibody producing memory B cells. Greater than 75independent B cell clones to the glycoproteins from different patientswere isolated. Binding and specificity profiles of LASV GPCcomponent-specific huMAbs were determined in immunoprecipitation andELISA assays.

LASV plaque reduction neutralization test (PRNT) assay. Lassa virus(Josiah, GA391, and 803213 strains, for which good guinea pig modelsexist and are available) may be pre-incubated with various dilutions(e.g., about 10 pM to about 300 nM) of each MAb prior to infection ofVero or Vero E-6 cells. Virus may be removed after infection by washingtwice with phosphate-buffered saline (PBS) and cell medium with 0.5%agarose overlay may be added to each culture. Plaques may be countedabout 48 hours thereafter following neutral red staining. The level ofinhibition is then plotted against concentration and an IC50 (amount ofprotein required to block 50% entry) can be calculated.

Two identified LASV huMAbs designated as GP10.4B and GP19.7E displayedvirus neutralization in vitro in the LASV plaque reductionneutralization test (PRNT) assay. GP19.7E was significantly more potentthan 10.4B. The huMAbs GP10.4B and GP19.7E also exhibited significantneutralization potential against live LASV. The heavy chain (HC) andlight chain (LC) nucleotide sequences of GP10.4B and GP19.7E are shownin FIG. 3 (HC) and FIG. 4 (LC). The corresponding amino acid sequencesare shown in FIG. 5.

EXAMPLE 2 Preparation of Immunoprotective Primary MSC Expressing anAnti-LASV Immunoreactive Polypeptide

Adipose tissue-derived MSC were seeded in 6-well plates at a density ofabout 1 million cells/well in modified Eagle's medium alpha (MEM alpha)medium supplemented with 10% FBS. The following day, cells weretransfected with either LIPOFECTAMINE 2000 (Invitrogen) or PEI(Polyplus) and a pCMVintA_17HSD:huMAb 19.7E construct according tomanufacturer recommendations: Light and Heavy chain antibody genes fromhuMAbs GP19.7E was re-engineered with optimal Kozak sequences anddeconvolved 5′ UTRs, and cloned in a bicistronic mammalian expressionvector (FIG. 3, Panel B), in tandem and in opposing orientations. Intransiently transfected HEK-293T/17 cells, the opposing orientation geneconstructs resulted in higher secreted antibody levels than from tandemcounterparts. An NS0 cell line expressing huMAb GP19.7E was generated bytransfection with opposing antibody gene constructs. About 48 hours posttransfection, supernatants were harvested and serially diluted in1×PBS/0.1% BSA/0.1% TWEEN-20 for ELISA. Using this method the adiposeMSC produced about 60 ng/mL of GP19.7E antibody, versus undetectablesignal for an empty vector control.

EXAMPLE 3 LASV Protective Immunity Via Administration of MultipleNeutralizing Anti-LASV Antibodies

To demonstrate the immunotherapeutic activity of anti-LASV IgG huMAbs,outbred guinea pigs were injected with a single dose of approximately 30mg/Kg and 15 mg/Kg of MAb GP19.7E and MAb GP10.4B, respectively, on thesame day as LASV challenge. LASV Josiah was adapted to outbred guineapigs resulting in a uniformly lethal model by the intraperitoneal (i.p.)route. These outbred guinea pigs displayed clinical signs of the diseasesimilar to those observed in the inbred guinea pigs strain 13 andhumans. All control guinea pigs injected with antibody-free diluentsuccumbed with typical signs of Lassa fever by day 16 of the experiment(FIG. 6). The huMAb-treated guinea pigs were followed to 21 days. Noneof these huMAb-treated animals died or showed any signs of Lassa fever.These results demonstrate that treatment with this combination of Lassavirus glycoprotein specific huMAbs did not merely prolong survival, butprovided complete protection from the lethal effects of Lassa virus.

Preferred embodiments of this invention are described herein, includingthe best mode known to the inventors for carrying out the invention.Variations of those preferred embodiments may become apparent to thoseof ordinary skill in the art upon reading the foregoing description. Theinventors expect skilled artisans to employ such variations asappropriate, and the inventors intend for the invention to be practicedotherwise than as specifically described herein. Accordingly, thisinvention includes all modifications and equivalents of the subjectmatter recited in the claims appended hereto as permitted by applicablelaw. Moreover, any combination of the above-described elements in allpossible variations thereof is encompassed by the invention unlessotherwise indicated herein or otherwise clearly contradicted by context.

REFERENCES

The following references and any other previously identified referencesnot specifically listed below are incorporated herein by reference intheir entirety.

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We claim:
 1. Immunoprotective primary mesenchymal stems cells (IP-MSC)that episomally express immunoreactive polypeptides that specificallytarget a viral pathogen; the IP-MSC being adipose-derived mesenchymalstem cells transfected with one or more episomal vectors encodingexpressible immunoreactive polypeptides that specifically target thepathogen; wherein the immunoreactive polypeptides comprise amino acidsequences from an antigen-binding region of a neutralizing antibodyspecific for the pathogen; the antigen-binding region amino acidsequences being arranged to specifically bind to and neutralize thepathogen; wherein the viral pathogen is selected from the groupconsisting of: an adenovirus, a papillomavirus, a hepadnavirus, aparvovirus, a pox virus, Epstein-Barr virus (EBV), cytomegalovirus(CMV), a herpes simplex virus, roseolovirus, varicella zoster virus, afilovirus, a paramyxovirus, an orthomyxovirus, a rhabdovirus, anarenavirus, a coronavirus, a human enterovirus, hepatitis A virus, ahuman rhinovirus, polio virus, a retrovirus, a rotavirus, a flavivirus,a hepacivirus, and rubella virus; the episomal vector includes aninducible apoptosis gene; the immunoreactive polypeptides comprise thecomplementarity determining regions of the antibody; and the one or moreepisomal vectors are non-infective, non-integrative, circular episomalvectors.
 2. The IP-MSC of claim 1, wherein the IP-MSC also express oneor more other immunomodulating agents.
 3. The IP-MSC of claim 2, whereinthe one or more immunomodulating agents are selected from interleukinsand interferons.
 4. The IP-MSC of claim 3, wherein the one or moreimmunomodulating agents are selected from IL-2, IL-4, IL-6, IL-7, IL-9,IL-12, IFNα, IFNβ, and IFNω.
 5. The IP-MSC of claim 1, wherein theimmunoreactive polypeptides are selected from a full-length antibody, anantibody single-chain variable antibody fragment (ScFV), a monovalentantibody antigen-binding fragment (Fab), a divalent antibodyantigen-binding fragment (F(ab′)₂), a diabody, and a tribody.
 6. TheIPMSC of claim 1, wherein the one or more episomal vectors also encode atetracycline-controlled transcriptional activation system for activatingthe inducible apoptosis gene.
 7. A pharmaceutical composition fortreating or preventing an infection caused by the pathogen, thecomposition comprising the IP-MSC of claim 1 in a pharmaceuticallyacceptable carrier.
 8. The composition of claim 7, wherein theimmunoreactive polypeptides are selected from a full-length antibody, anantibody single-chain variable antibody fragment (ScFV), a monovalentantibody antigen-binding fragment (Fab), a divalent antibodyantigen-binding fragment (F(ab′)₂), a diabody, and a tribody.
 9. Amethod for treating or preventing an infection caused by the pathogen,the method comprising administering to a subject an effective dosage ofthe IP-MSC of claim
 1. 10. The method of claim 9, wherein theimmunoreactive polypeptides are selected from a full-length antibody, anantibody single-chain variable antibody fragment (ScFV), a monovalentantibody antigen-binding fragment (Fab), a divalent antibodyantigen-binding fragment (F(ab′)₂), a diabody, and a tribody. 11.Immunoprotective primary mesenchymal stems cells (IP-MSC) thatepisomally express immunoreactive polypeptides that specifically targetcytomegalovirus (CMV); the IP-MSC being adipose-derived mesenchymal stemcells transfected with one or more episomal vectors encoding expressibleimmunoreactive polypeptides that specifically target CMV; wherein theimmunoreactive polypeptides comprise amino acid sequences from anantigen-binding region of a neutralizing antibody specific for CMV; theantigen-binding region amino acid sequences being arranged tospecifically bind to and neutralize CMV; wherein the episomal vectorincludes an inducible apoptosis gene; the immunoreactive polypeptidescomprise the complementarity determining regions of the antibody; andthe one or more episomal vectors are non-infective, non-integrative,circular episomal vectors.
 12. The IP-MSC of claim 11, wherein theIP-MSC also express one or more other immunomodulating agents.
 13. TheIP-MSC of claim 12, wherein the one or more immunomodulating agents areselected from interleukins and interferons.
 14. The IP-MSC of claim 13,wherein the one or more immunomodulating agents are selected from IL-2,IL-4, IL-6, IL-7, IL-9, IL-12, IFNα, IFNβ, and IFNω.
 15. The IP-MSC ofclaim 11, wherein the immunoreactive polypeptides are selected from afull-length antibody, an antibody single-chain variable antibodyfragment (ScFV), a monovalent antibody antigen-binding fragment (Fab), adivalent antibody antigen-binding fragment (F(ab′)₂), a diabody, and atribody.
 16. The IPMSC of claim 11, wherein the one or more episomalvectors also encode a tetracycline-controlled transcriptional activationsystem for activating the inducible apoptosis gene.
 17. A pharmaceuticalcomposition for treating or preventing a CMV infection, the compositioncomprising the IP-MSC of claim 11 in a pharmaceutically acceptablecarrier.
 18. The composition of claim 17, wherein the immunoreactivepolypeptides are selected from a full-length antibody, an antibodysingle-chain variable antibody fragment (ScFV), a monovalent antibodyantigen-binding fragment (Fab), a divalent antibody antigen-bindingfragment (F(ab′)₂), a diabody, and a tribody.
 19. A method for treatingor preventing a CMV infection, the method comprising administering to asubject an effective dosage of the IP-MSC of claim
 11. 20. The method ofclaim 19, wherein the immunoreactive polypeptides are selected from afull-length antibody, an antibody single-chain variable antibodyfragment (ScFV), a monovalent antibody antigen-binding fragment (Fab), adivalent antibody antigen-binding fragment (F(ab′)₂), a diabody, and atribody.